POS0240 EFFECT OF METHOTREXATE UPON OXIDATIVE STRESS MEDIATED TRANS-DIFFERENTIATION OF NEUTROPHILS IN RHEUMATOID ARTHRITIS
Authors: A. Bagchi, P. Ghosh, A. Ghosh, M. Chatterjee
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Rheumatoid arthritis (RA), a chronic systemic autoimmune disorder, is associated with inflammation coupled with oxidative stress, and translates into infiltration of immune cells in inflamed joints i.e. predominantly neutrophils (~90%). In previous studies, a subpopulation of accumulated neutrophils trans-differentiated into neutrophil-dendritic cell hybrids or N-DCs (CD66b<sup>+</sup>/CD83<sup>+</sup>). This N-DCs acquired antigen presenting properties (HLA-DR<sup>+</sup>/CD80<sup>+</sup>/CD86<sup>+</sup>) and generated enhanced reactive oxygen species (ROS), collectively causing oxidative damage in the synovial fluid (SF) [<b>1,2</b>]. The impact of Methotrexate (MTX) upon redox status along with trans-differentiation and degree of apoptosis in neutrophils remains unclear and was the focus of this study. <h3>Objectives</h3> As oxidative stress is known to facilitate trans-differentiation of neutrophils, a critical factor for disease progression, the impact of MTX was studied on the status of trans-differentiation i.e. CD83<sup>+</sup>/HLA-DR<sup>+</sup> in neutrophils sourced from healthy controls along with their status of activation, generation of ROS and degree of apoptosis. <h3>Methods</h3> In the SF of RA cases (n=25), frequency of trans-differentiation was examined by immunophenotyping (CD66b, CD83, HLA-DR). The <i>ex-vivo</i> effect of MTX on neutrophils (+/- Phorbol Myristate Acetate or PMA; to mimic the redox microenvironment in SF of RA) sourced from healthy controls was studied in terms of their status of trans-differentiation (CD66b<sup>+</sup>/CD83<sup>+</sup>/HLA-DR<sup>+</sup>), activation (Myeloperoxidase or MPO<sup>+</sup>), generation of ROS using CMH<sub>2</sub>DCFDA and proportion of apoptosis (Annexin V<sup>+</sup>, pro-apoptotic Caspase 3<sup>+</sup>, anti-apoptotic Bcl-2<sup>+</sup>) in flow cytometer. <h3>Results</h3> In the SF of RA cases, frequency of trans-differentiation (CD66b<sup>+</sup>/CD83<sup>+</sup>/HLA-DR<sup>+</sup>) correlated positively with DAS28-CRP (r= 0.70, p&lt;0.001), culminating in their disease promoting role. MTX (at non-toxic concentrations, 0-100 μM) did not impact on generation of ROS in neutrophils sourced from healthy controls. However, when oxidative stress was induced by PMA, MTX in a dose dependent fashion synergistically facilitated a redox imbalance (enhanced frequency of ROS<sup>high</sup> neutrophils) coupled with enhanced trans-differentiation (CD83<sup>high</sup>), antigen presentation (HLA-DR<sup>high</sup>), activation (%MPO<sup>+</sup>) and apoptosis i.e increased Annexin V<sup>+</sup>, Caspase 3<sup>+</sup> along with compromised Bcl-2. <h3>Conclusion</h3> Oxidative stress can trigger trans-differentiation of neutrophils, which is a crucial for disease progression, and preventing trans-differentiation can be a potential therapeutic target in RA. Although MTX, an immunomdulator is the drug of choice for treatment of RA, our studies exert a certain degree of caution, as MTX in an enhanced pro-oxidant milieu facilitated generation of oxidative stress, and could impact on disease progression. <h3>Reference</h3> [1]<b>Bagchi A</b>, Ghosh P, Ghosh A, Chatterjee M. Role of oxidative stress in induction of trans-differentiation of neutrophils in patients with rheumatoid arthritis. Free Radical Research. 2022 3;56(3-4):290-302. [2]Iking-Konert C, Ostendorf B, Sander O, Jost M, Wagner C, Joosten L, Schneider M, Hänsch GM. Transdifferentiation of polymorphonuclear neutrophils to dendritic-like cells at the site of inflammation in rheumatoid arthritis: evidence for activation by T cells. Annals of the rheumatic diseases. 2005 1;64(10):1436-42. <h3>Acknowledgements</h3> I want to acknowledge Prof. Mitali Chatterjee for constantly guiding and supporting in my Ph.D work. I also want to thank Prof. Alakendu Ghosh and Prof. Parasar Ghosh for providing biological samples required for this study. Furthermore, I have special regards to "Multidisciplinary Research Unit' in IPGMER for providing instruments and ICMR-Govt. of India for my Senior Research Fellowship. <h3>Disclosure of Interests</h3> None Declared.